Radical SAM enzymes are a remarkably diverse enzyme superfamily with more than 60,000 members distributed throughout all kingdoms of life. The versatility of radical SAM chemistry is unique in biology, with a single type of initiation process - involving the reductive cleavage of SAM by an iron-sulfur cluster to generate a reactive radical intermediate - ultimately responsible for reactions as diverse as C-C bond cleavage, sulfur insertion into C-H bonds, simple or complex rearrangements, and formation of thioether crosslinks. The long-term objective of this project is to provide a detailed, molecular-level understanding of the mechanistic steps common to radical SAM enzymes; such understanding will be essential in order to exploit these enzymes for the benefit of public health. At least nine distinct radical SAM enzymes have been identified in humans, including enzymes involved in lipoyl cofactor biosynthesis and in the antiviral response; several other human radical SAM enzymes have not yet been functionally characterized. In addition, radical SAM enzymes are prevalent in microbes, including microbes that cause human illness and those that are beneficial. The specific aims of the current proposal are: 1) to identify and characterize radical SAM reaction intermediates; 2) to utilize SAM analogs to probe radical SAM mechanisms; 3) to elucidate the nature and role of the essential monovalent cation in the radical SAM enzyme pyruvate formate- lyase activating enzyme; and 4) to examine the origin and functional significance of valence localization. The experimental plan will involve isolating radicl intermediates using one of three approaches: rapid freeze-quench, cryoreduction, or stabilizing analogs of radical intermediates using appropriately designed substrate or cofactor analogs. Site-specific incorporation of NMR-active nuclei will allow for characterization of radical intermediates using electron-nuclear double resonance. The nature and significance of the monovalent cation site will be probed by a combination of electron paramagnetic resonance, cation substitution, spectroelectrochemical titrations, and site-directed mutagenesis. Valence localization will be studied through a combination of electron paramagnetic resonance, electron-nuclear double resonance, Mssbauer spectroscopy, and site-directed mutagenesis. Together, these studies will provide fundamental new insights into the common chemical mechanism employed by this ubiquitous and diverse enzyme superfamily.